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Background: Persons dually diagnosed with opioid and cocaine dependence (OD + CD) present a clinical challenge and are at risk of morbidity and mortality. The time of escalation of heroin and cocaine exposure in persons with OD + CD remain understudied, and the influence of gender and other variables have not been examined. This observational study focused on the time of escalation of heroin and cocaine in volunteers with OD + CD, examining gender and exposure to other drugs (e.g., cannabis or alcohol) as predictors. Ages of first use and of onset of heaviest use of each drug were collected (in whole years). Time of escalation was defined as the interval between age of first use and onset of heaviest use. Volunteers: sequentially ascertained adult volunteers recruited from the New York Metropolitan area, of which n = 297 were diagnosed with OD + CD. Methods: Instruments administered were the SCID-I diagnostic interview (DSM-IV criteria), BIS-11 impulsiveness scale, and KMSK scales, dimensional measures of maximal exposure to specific drugs. Results: In volunteers with OD + CD, ages of onset of heaviest use of cannabis (median age = 15) and alcohol (median age = 19) were in adolescence or emerging adulthood and preceded those for heroin and cocaine (median ages = 26 and 25, respectively). Maximal levels of cannabis and alcohol exposure were high, in volunteers with OD + CD. In adjusted Cox regressions, gender was not a significant predictor of time of heroin or cocaine escalation. However, more rapid time of alcohol escalation was a predictor of more rapid time of escalation of both heroin and cocaine, in volunteers with OD + CD.

Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-alpha) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis. IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.

Linked Article: Klassen et al. Br J Dermatol 2019; 181:1207-1215.

APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminasedependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNAbinding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination. IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 265 assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

CD4(+) T cell help is required for the generation of CD8(+) cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4(+) T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (T-CM) cells. More importantly, help signals regulate the size and function of the effector memory T (T-EM ) cell pool. Helped T-EM cells produce Granzyme B and IFN gamma upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4(+) T cell help optimizes CTL memory by creating T-EM cells with innate and helpindependent antigen-specific recall capacities.

Examining the links between neuronal activity, transcriptional output, and synaptic function offers unique insights into how neurons adapt to changing environments and form memories. Epigenetic markers, such as DNA methylation and histone modifications, have been implicated in the formation of not only cellular memories such as cell fate, but also memories of experience at the organismal level. Here, we review recent advances in chromatin regulation that contribute to synaptic plasticity and drive adaptive behaviors through dynamic and precise regulation of transcription output in neurons. We discuss chromatin-associated proteins, histone variant proteins, the contribution of cis-regulatory elements and their interaction with histone modifications, and how these mechanisms are integrated into distinct behavior and environmental response paradigms.

Alcohol relapse is a treatment goal for alcohol dependence and the target for medications' development. Clinically utilized nalfurafine (NFF) is a potent and selective kappa- opioid receptor (KOP-r) agonist, with fewer side effects (e.g., sedation or anhedonia) than classic KOP-r full agonists. We have recently found that NFF reduces excessive alcohol drinking in mice via a KOP-r-mediated mechanism. Here, we further investigated whether NFF alone (1-10 mu g/kg) or in combination with naltrexone (NTX, mu-opioid receptor [MOP-r] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. Nalmefene (NMF, clinically utilized KOP-r partial agonist with MOP-r antagonism) was used as a reference compound for the effects on mouse ADE of new NFF + NTX combination. After exposed to 3-week intermittent- access alcohol drinking (two-bottle choice, 24-h access every other day), both male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. NFF prevented the ADE in a dose-dependent manner in both male and female mice. A combination of NFF with NTX reduced the ADE without sex differences at doses lower than those individual effective ones, suggesting synergistic effects between the two compounds. NMF prevented the ADE in both sexes, while selective KOP-r antagonist nor-BNI had no effect. Our new study suggests that a combination of clinically-utilized, potent KOP-r agonist NFF with low-dose NTX has therapeutic potential in alcohol "relapse" treatment.